Real-time fluorescence imaging platform for high-throughput screening of arbuscular mycorrhizal fungi enhancing plant nutrient uptake

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They enhance host nutrient and water acquisition in exchange for photosynthetic carbon. Current AMF research relies on field trials, compartmented cultivation and pot cultures‒methods that are time-consuming (months to years) and unable to monitor dynamic nutrient transport, thus limiting efficient strain screening. Results We developed a real-time fluorescence imaging platform integrating sterile symbiotic microchambers with photodiode array detection. This system enables non-invasive, quantitative tracking of nutrient flux at plant-fungal interface. Distinct AMF strains exhibit significant differences in fluorescence kinetics—such as accumulation rate and peak intensity—providing measurable indicators of transport efficiency. The platform allows high-throughput functional screening of AMF strains, dramatically accelerating the identification of high-performance symbionts. Conclusion Our method overcome the temporal and technical limitations of conventional AMF screening approaches. By enabling simultaneous real-time monitoring and high-throughput analysis, it shortens screening cycles and establishes a standardized framework for (1) precision breeding of efficiency AMF strains, (2) mechanistic study of nutrient exchange, and (3) development of sustainable microbial inoculants. arbuscular mycorrhizal fungi nutrient transport cytoplasmic streaming high-throughput screening fluorescence imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Arbuscular mycorrhizal fungi (AMF) form mutualistic symbioses with approximately 80% of land plant species, playing a crucial role in enhancing host mineral nutrient acquisition [ 1 , 2 ] . This obligate symbiotic relationship is based on a bidirectional exchange: plants provide carbon sources such as fatty acids and sugars to the fungi, while AMF extend the root system via their hyphae networks [ 3 , 4 ] and secrete metabolites including proteases and siderophores, which facilitate the mobilization of organic nitrogen and metallic nutrients [ 5 , 6 ] . Moreover, AMF hyphae recruit beneficial rhizosphere microbes (e.g., Pseudomonas, Bacillus) through carbon-rich exudates, establishing synergistic interactions that further improve plant uptake of phosphorus, nitrogen, and micronutrients [ 7 – 11 ] . The primary site of nutrient exchange is the arbuscule—a highly branched structure formed within root cortical cells that greatly expands the symbiotic interface [ 12 – 14 ] . Efficient nutrient transport relies on cytoplasmic streaming within the hyphae, which enables both intrahyphal diffusion and long-distance translocation of nutrients [ 15 – 18 ] Upon arrival at the arbuscules, nutrients are released across the periarbuscular membrane for plant assimilation [ 19 ] , underscoring the fundamental role of cytoplasmic streaming in symbiotic efficiency [ 20 ] . Conventional methods for studying AMF symbiosis, such as field trials and compartmented cultivation systems, face significant limitations. These approaches typically depend on endpoint measurements of nutrient concentrations in plant tissues, thereby failing to capture the dynamic nature of nutrient transport processes [ 21 ] . Moreover, non-axenic conditions increase the risk of microbial contamination, and soil variability often compromises experimental reproducibility [ 22 , 23 ] . For perennial plants, these methods can require months to years to yield results, greatly restricting the capacity for high-throughput strain screening. To address these constra challenges, we developed micro-chamber system integrated with real-time fluorescence monitoring. This platform allows for: (1) sterile co-culture, eliminating microbial contamination; (2) in situ tracking of nutrient fluxes using fluorescent tracers; and (3) quantitative analysis of uptake kinetics through spatiotemporal fluorescence profiling. Beyond providing mechanistic insights into AMF-mediated nutrient acquisition, this system offers a standardized framework for the high-throughput phenotyping of AMF strains with superior transport efficiency. This advancement supports ongoing efforts to improve crop nutrient-use efficiency and promote sustainable agriculture practices [ 4 , 24 ] . Materials and Methods Plant Material and Hairy Root Induction To ensure consistent biomass production，transgenic hairy roots were induced in carrot ( Daucus carota L.) explants using Agrobacterium rhizogenes strain ACCC10060 [25] . These axenic hairy roots were subsequently co-cultivated with surface-sterilized AMF spores to establish symbiotic samples. The procedure involved the following steps: Preparation of Agrobacterium rhizogenes Inoculum: (i) A single bacterial colony was inoculated into YEB broth (Yeast Extract Broth; Coolaber, Beijing, China) and cultured at 28°C with agitation 200 rpm until an OD 600 of 1.0 was reached. (ii) A 2 mL aliquot of this pre-culture was transferred to 100 mL of fresh YEB broth and incubated under identical the same conditions until the OD 600 reached 0.6. (iii) Spores were pelleted by centrifugation at 3,000 × g for 3 min at 25°C. (iv) The pellet was resuspended in MS liquid medium supplemented with 100 μM acetosyringone (Solarbio, Beijing, China ; dissolved in DMSO) to prepare the inoculum. Hairy Root Induction: Carrot ( Daucus carota L.) taproots were subjected to sequential surface sterilization as follows: (i) Rinsing with distilled water. (ii) Removal of epidermal tissues. (iii) Immersion in 75% (v/v) ethanol for 30 seconds. (iv) Treatment with 1% (v/v) sodium hypochlorite for 10–20 minutes. Co-culture: Explants were co-cultured for 3 days in darkness at 25–28°C. Decontamination: Using Medium I (1% [w/v] agar supplemented with 400 mg/L Timentin; Coolaber, Beijing, China). Subculture: Roots were maintained on hormone-free full-strength MS medium (hopebiol, Qingdao, China) at 26 ± 1°C. AM Fungal Spore Isolation and Sterilization: Individual spores of Glomus versiforme and Rhizophagus irregularis were isolated from soil samples using a micropipette puller (Model P-97, Sutter Instrument, Novato, CA, USA). Surface sterilization was performed as follows: 1. Ultrasonic cleaning: 40 kHz, 100 W, for 2 minutes in sterile water (KQ-3200DE, Kunshan Ultrasonic Instruments, Jiangsu, China). 2. Chemical sterilization: 10 minutes in Solution A (2% [w/v] chloramine-T (Coolaber, Beijing, China) + 0.01% [v/v] Tween-20 (Coolaber, Beijing, China)). 3. Antibiotic treatment: 10 minutes in Solution B (200 μg/mL streptomycin + 100 μg/mL gentamicin (both from Solarbio, Beijing, China)). Sterilized spores were rinsed five times with sterile water prior to inoculation. Symbiotic Co-culture Approximately 20 surface-sterilized spores were inoculated 1–2 cm from carrot hairy roots in modified Strullu-Romand (MSR) agar medium [26] . Chambers were sealed and incubated at 25–28°C in darkness. Symbiosis was confirmed at 40–60 days post-inoculation (dpi) by observing hyphal penetration under an Olympus BX63 microscope (Olympus, Tokyo, Japan). Symbiotic Micro-chamber Construction: A sealed microfluidic culture chamber was constructed using quartz glass (Fig. 3), consisting of custom-made hollow quartz plates and cover slips. The chamber has overall external dimensions of 70 × 70 × 5 mm (L × W × H) and an internal cavity measuring 40 × 40 × 5 mm. Two centrally located alignment grooves (1 × 1 × 5 mm) along opposite edges of the cavity allow precise compartmentalization. A sterile nylon membrane (30 µm pore size; UV-treated and permeable only to AMF hyphae) divides the chamber into separate plant and hyphal compartments. Cover slips (50 × 50 mm) were sealed to the chamber base using high-vacuum silicone grease (Dow Corning, Midland, MI, USA), followed by autoclave sterilization (121°C, 30 min, 15 psi) to ensure sterility. Fluorescence Detection System: Detailed circuit design and noise analysis are provided in Supplementary Fig. S1 A high-sensitivity transimpedance amplifier (TIA) circuit (Fig.4) was developed for detecting weak bioluminescent signals. The system consists of three optimized subsystems: 1. Power Filtering: A symmetrical power filtering network using parallel 100 nF ceramic capacitors (X7R) and 10 μF tantalum capacitors per supply rail (±12.5 V to ±18 V) to suppress multi-frequency power noise, achieving μV-level ripple essential for nanoampere-level signal integrity. 2. Photodiode: A custom photodiode operating at zero reverse bias (Vr = 0 V) with a junction capacitance (Cd) of 150 pF and shunt resistance (Rsh) of 600 MΩ, eliminating dark current-induced shot noise while maximizing photon collection area for a peak photocurrent (Ip) of 100 nA. 3. TIA Core: A single-stage TIA utilizing an AD795 operational amplifier with ultra-low input voltage noise (1.2 nV/√Hz). A 100 MΩ thin-film feedback resistor (Rf) provides a gain of 10⁸ V/A, A strategically selected 1.3 pF compensation capacitor (Cf) stabilizes the high-impedance node by damping the resonance peak (Q = 0.453), extending the bandwidth to 3.26 kHz—significantly beyond the theoretical limit of 1/(2πRfCd) ≈ 10.6 Hz—without compromising the 190 μs rise time. The total output noise is 164 μV RMS (1.64 pA RMS referred to input), predominantly contributed by the op-amp’s voltage noise (94.5%). The design achieves an SNR of 86.6 dB (ENOB = 14.1 bits), enabling reliable detection of sub-picoampere bioluminescent signals within the target bandwidth of 1 kHz. Optoelectronic assembly ： The fluorescence detection module (Fig. 5) integrates four components: an X-Cite 120Q excitation lamp (Exelitas Technologies), an excitation filter (Ex-filter; #86-977 , Edmund), an emission filter (Em-filter; #86-979 , Edmund), and a photodiode(S1336-44BQ, Hamamatsu Photonics, Hamamatsu, Japan); data acquisition was performed using an NI USB-6210 interface (National Instruments, Austin, TX, USA). In this configuration, the excitation source and Ex-filter are positioned above the plant compartment, while the Em-filter and photodiode are optically aligned below. The excitation source delivers targeted illumination to the plant samples.When fluorophore-labeled nutrients enter the detection zone, they emit fluorescence upon excitation. The emitted light passes through the Em-filter to removal stray light before being converted into photocurrent by the photodiode. Fluorescence Detection Procedure: Carrot hairy roots cultured in-house and surface-sterilized AMF spores were used in the experiments. Hairy roots were inoculated in the plant compartment and spores in the hyphal compartment, both containing modified Strullu-Romand (MSR) medium. Sealed microchambers were incubated in darkness at 25-28°C. After AMF hyphal germination and penetration through the nylon membrane to establish symbiosis, co-culture continued for 40-60 days until extensive hyphal networks developed. The upper lid was gently removed, and FM4-64 fluorescent dye was applied to the hyphal compartment to label hyphae for cytoplasmic streaming quantification. The root-hyphal junction was positioned over the photodiode detector and enclosed within the detection module. Excitation was then initiated for real-time monitoring of fluorescence dynamics. Data Acquisition and Analysis The photodiode converted low-amplitude fluorescence signals into current signals. Which were processed by a transimpedance amplifier (TIA) and filter, yielding analog voltage signals. These were digitized at 250 kS/s using an via an NI USB-6210 data acquisition interface (National Instruments). Raw data were collected with a custom LabVIEW program. Data acquisition was performed at 250 kS/s using LabVIEW 2023 (National Instruments). Fluorescence kinetics were analyzed using custom MATLAB scripts (MathWorks, Natick, MA, USA). Statistical Analysis and Reproducibility Data are presented as mean ± SD of three biological replicates. Significance was determined using two-tailed t-test ( p < 0.05, p < 0.01) . Results Real-Time Visualization of Cytoplasmic Streaming Dynamics Extraradical hyphae of arbuscular mycorrhizal fungi (AMF) form extensive networks that facilitate nutrient acquisition through cytoplasmic streaming to arbuscules [27] . However, in situ monitoring of nutrient flux remains challenging due to soil heterogeneity. To address this, we developed a real-time imaging platform using the membrane-selective fluorophore FM4-64 (10 μM; Fig. 6). This dye effectively labeled live root tissues, intraradical hyphae, and spores within sterile micro-chambers. Using fluorescence microscopy (Olympus BX63; 560/640 nm, 5 fps), we quantified directional transport of fluorescent signals, establishing cytoplasmic streaming as a direct proxy for nutrient flux. Strain-Specific Kinetics of Cytoplasmic Streaming Our imaging platform tracked the transport of FM4-64 from distal hyphal tips (applied 1 cm from detection zones) to symbiotic interfaces (Fig. 7). Quantitative analysis revealed distinct kinetics profiles between AMF strains: Glomus versiforme exhibited rapid fluorescence accumulation (200–500 s: slope = 0.01506 ± 0.0008ΔF/F₀·s⁻¹, p < 0.01), reaching a plateau at 500 s ( peak ΔF/F₀ = 0.0007 ± 0.0027). Rhizophagus irregularis showed a sustained increases until 800 s ( slope = 0.0052 ± 0.0003 ΔF/F₀·s⁻¹), with a lower peak intensity (ΔF/F₀ = 0.0014 ± 0.0007; p = 0.008 vs. G. versiforme ; Fig. 7C).These findings indicate efficient tracer delivery to symbiotic regions, with G . versiforme demonstrating superior transport kinetics. Quantitative Comparison of Hyphal Transport Capacity We quantified interspecific variation by analyzing fluorescence dynamics over a 12 minute period (Fig. 8): G. versiforme achieved a 2.3-fold higher initial transport rate (3–6 min: 3.131 ± 0.864 vs. 1.311 ± 0.305 ΔF/F₀·min⁻¹ in R. irregularis , p < 0.05) and reached peak intensity at 9 min (ΔF/F₀ = 4.645 ± 0.465). R. irregularis exhibited a delayed peak at 12 min (ΔF/F₀ = 4.029 ± 0.238; not significant vs. G. versiforme after 9 min; Fig. 8). Statistical analysis (n = 3 biological replicates) confirmed the superior transport capacity of G. versiforme (p < 0.05 at 6 and 9 min), highlighting functional divergence in AMF symbiotic efficiency. Discussion Our compartmentalized microchamber system overcomes the limitations of traditional pot cultures by enabling real-time monitoring of nutrient dynamics at the hypha-root interface (Fig. 1 ). Key advances include: (1) Sterility-controlled co-culture , which eliminates artifacts from soil sterilization and ensures specific colonization by AMF; (2) Integrated optoelectronic sensors that capture continuous nutrient fluxes, revealing kinetic bottlenecks undetectable with endpoint assays. Although this platform allows precise manipulation of plant-fungal interactions, its current simplified representation of the rhizosphere microbiome does not account for essential cross-kingdom interactions. For example, bacterial communities influenced by AMF-derived strigolactones may enhance symbiosis efficiency through auxin signaling or siderophore production. Future designs incorporating modular bacterial chambers will be crucial for elucidating such tripartite interactions [ 28 – 30 ] . Constraints in Probe diversity also limit the scope of detection. Existing fluorescent target only major minerals (e.g., Pi, NH₄⁺), leaving trace elements (e.g., Zn²⁺, Cu²⁺) unmonitored. Integrating dynamic flux measurements with multi-omics approaches—such as spatially resolved metabolomics of arbusculated cells—could uncover mechanisms governing nutrient exchange across biological scales [ 31 – 34 ] . To bridge the gap between laboratory and field conditions, we propose a tiered validation pipeline: (1) High-throughput screening of up to 200 AMF strains per experiment using microchambers; (2) Controlled-environment validation with defined substrates that simulate soil heterogeneity; (3) Field assessment via rhizoboxes to quantify functional stability under realistic agricultural conditions (Fig. 3 ). This framework balances screening efficiency with ecological relevance, accelerating the development of beneficial AMF strains into sustainable inoculants. In summary, our system provides a transformative platform for analyzing arbuscular mycorrhizal symbiosis dynamics. By combining real-time sensing with scalable validation, it facilitates the translation of AMF research from mechanistic insights to practical applications, supporting advances in microbiome engineering for sustainable agriculture. Conclusions We developed a real-time fluorescence imaging platform incorporating photodiode arrays to dynamically quantify nutrient flux at plant-AMF symbiotic interfaces. This system enables high-throughput functional classification of arbuscular mycorrhizal fungi (AMF) strains by monitoring fluorescence kinetics—such as FM4-64 tracer accumulation rates—achieving a tenfold increase in screening speed compared to conventional field-based methods. Our approach effectively connects mechanistic research with sustainable agriculture, promoting targeted AMF breeding and microbiome engineering. Abbreviations AMF Arbuscular mycorrhizal fungi YEB Yeast Extract Broth MSR Strullu-Romand medium TIA transimpedance amplifier Declarations Funding This work was supported by the China Huanghuai Sea High-yield Key Cultivation Technology Innovation and Integration Demonstration (Grant No. 2023YFD2300204), the National Natural Science Foundation of China (Grant No. 32401739) and the China National Modern Agricultural Production Technology System(CARS-03). Author information Authors and Affiliations 1. Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Zhengzhou 450046, Henan, China 2. National Engineering Research Centre for Wheat, Zhengzhou 450046, Henan, China Yonghua Wang, Bo Yu, Tiancai Guo, GuangLe Zhang, Lixue Yuan, Yongxin Zhang, Xinyuan Zhang, Li Zhang, Ruxue Chen 3. College of Resources and Environment, Henan Agricultural University, Zhengzhou 450046, Henan, China Zhuangzhuang Wang 4. Department of Crop and Soil Sciences of North Carolina State University, Raleigh, 27695, North Carolina, US Xiaohang Wang Contributions B. Y. and G.L. Z. designed and fabricated the circuit board, collected and analyzed data, and drafted the manuscript. B. Y., Y.H. W. and T.C. G. conceived and designed the study, developed the methodology, and revised the manuscript substantively. L.X. Y. and Y.X. Z. contributed to data acquisition, analysis, and interpretation; X.H. W., X. Y. Z., L. Z., R.X. C., and Z.Z. W. contributed to data acquisition and collation; All authors read and approved the final manuscript and agree to be accountable for all aspects of the work. Corresponding authors Correspondence to Bo Yu and Yonghua Wang Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. 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Plant–microbiome interactions: from community assembly to plant health. Nat Rev Microbiol. 2020;18(11):607–21. https://doi.org/10.1038/s41579-020-0412-1 . Carper DL, Appidi MR, Mudbhari S, Shrestha HK, Hettich RL, Abraham PE. The Promises, Challenges, and Opportunities of Omics for Studying the Plant Holobiont. Microorganisms. 2022;10(10). https://doi.org/10.3390/microorganisms10102013 . Supplementary Figure Supplementary Figure S1 is not available with this version. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7568699","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":515031193,"identity":"a3d36661-6789-4d05-9036-b14b1f98154d","order_by":0,"name":"Guangle Zhang","email":"","orcid":"","institution":"Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping","correspondingAuthor":false,"prefix":"","firstName":"Guangle","middleName":"","lastName":"Zhang","suffix":""},{"id":515031194,"identity":"33667287-ed34-49ee-9dc2-02cd5c096b9f","order_by":1,"name":"Lixue 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University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohang","middleName":"","lastName":"Wang","suffix":""},{"id":515031197,"identity":"84ab7461-e9b7-4481-be8d-7670a3688683","order_by":4,"name":"Li Zhang","email":"","orcid":"","institution":"Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhang","suffix":""},{"id":515031198,"identity":"38300f8f-27a9-4557-abca-1d1dc6477c36","order_by":5,"name":"Xinyuan Zhang","email":"","orcid":"","institution":"Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping","correspondingAuthor":false,"prefix":"","firstName":"Xinyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":515031199,"identity":"8a7c4f88-6e95-4b52-95c3-229e6c819ab7","order_by":6,"name":"Tiancai Guo","email":"","orcid":"","institution":"Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping","correspondingAuthor":false,"prefix":"","firstName":"Tiancai","middleName":"","lastName":"Guo","suffix":""},{"id":515031200,"identity":"925d6403-6b76-4d95-8a1c-bdb4cd4768a6","order_by":7,"name":"Ruxue Chen","email":"","orcid":"","institution":"Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping","correspondingAuthor":false,"prefix":"","firstName":"Ruxue","middleName":"","lastName":"Chen","suffix":""},{"id":515031201,"identity":"33f112d5-6837-428b-aa55-7dc7767929d7","order_by":8,"name":"Zhuangzhuang Wang","email":"","orcid":"","institution":"College of Resources and Environment, Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhuangzhuang","middleName":"","lastName":"Wang","suffix":""},{"id":515031202,"identity":"f2dc8e3d-57e3-4bac-a8ac-20bd46239887","order_by":9,"name":"Bo Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYFACHgaGjw0INnFaGGeSrIWZlyQtBjdyDz623WGTxz8jgfHB2zYGeXPCWvKSjXPPpBVL3EhgNpzbxmC4s4Gglhwz6dy2w4kNNxLYpHnbGBIMDhCjxbLtf+L8Gwnsv4nXwth2IHED0BZmorRInnmXbNjblpy48czDZsk55yQMNxDSwnc89+CDn212ifOOJx/88KbMRp6gLQoIBYwNQEKCgHogkG8grGYUjIJRMApGOgAAwR1DDzrprfYAAAAASUVORK5CYII=","orcid":"","institution":"Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping","correspondingAuthor":true,"prefix":"","firstName":"Bo","middleName":"","lastName":"Yu","suffix":""},{"id":515031203,"identity":"362cad7b-2306-49c5-945f-2f5a3db98ced","order_by":10,"name":"Yonghua Wang","email":"","orcid":"","institution":"Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping","correspondingAuthor":false,"prefix":"","firstName":"Yonghua","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-09-09 03:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7568699/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7568699/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91963581,"identity":"188a7e2a-a1ad-4ecd-808d-e6c2cd889e65","added_by":"auto","created_at":"2025-09-23 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08:23:22","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106456,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/94f616d21ab3e4efadb58f84.html"},{"id":91964960,"identity":"64ff54ef-b600-47d4-bd85-09655e4be411","added_by":"auto","created_at":"2025-09-23 08:15:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":564058,"visible":true,"origin":"","legend":"\u003cp\u003eHairy roots induced in carrot using \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e strain ACCC10060\u003c/p\u003e\n\u003cp\u003e(A) Carrot explants 10 days post-infection; (B) Emergence of hairy roots at 15 days post-infection; (C) Extensive root development at 30 days. Scale bars: 1 cm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/5469ff2f952862423caed1ee.png"},{"id":91963565,"identity":"75a233a1-8bb6-47cf-a598-0fee1fd411c9","added_by":"auto","created_at":"2025-09-23 08:07:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":284002,"visible":true,"origin":"","legend":"\u003cp\u003eIsolation of individual AMF spores.\u003c/p\u003e\n\u003cp\u003e(A-D) Schematic diagram of the spore separation procedure; (E-H) Physical representation of individual spore separation. Scale bar: 40 µm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/730ea735599583a7cad24faa.png"},{"id":91963568,"identity":"be9261d0-fd07-486d-884c-7963227b1d80","added_by":"auto","created_at":"2025-09-23 08:07:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":727589,"visible":true,"origin":"","legend":"\u003cp\u003eCompartmentalized symbiotic micro-chamber.\u003c/p\u003e\n\u003cp\u003e(A) Schematic: (i) Root compartment, (ii) Hyphal compartment, (iii) Nylon membrane (30 μm pore).(B) Physical prototype. Scale bar: 1 cm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/b0d2350a49f30c2b45691ce0.png"},{"id":91963566,"identity":"b3a52c67-57c0-4b04-bb74-d5a1918dfefc","added_by":"auto","created_at":"2025-09-23 08:07:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":184384,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent signal acquisition board\u003c/p\u003e\n\u003cp\u003e(A) Circuit schematic: TIA with 100 MΩ feedback resistor; (B) Noise spectrum (bandwidth: 3.26 kHz); (C) Signal detection output.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/a40e8320b4a471ed0d1e7de0.png"},{"id":91964963,"identity":"6b1c6dfe-766d-4265-ab92-3084f532af5e","added_by":"auto","created_at":"2025-09-23 08:15:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":279599,"visible":true,"origin":"","legend":"\u003cp\u003eSpatiotemporal nutrient flux quantification system\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram; B: Physical setup\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/443111ef35a6eb541fc32a6f.png"},{"id":91965434,"identity":"55195ae7-e039-486d-909b-d7de4b897d40","added_by":"auto","created_at":"2025-09-23 08:23:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":426366,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent staining patterns of hyphae, roots, and spores\u003c/p\u003e\n\u003cp\u003e(B, C) Long arrows indicate spores and short arrows indicate hyphae before staining; (E, F) long arrows indicate fluorescently stained spores and short arrows indicate stained hyphae. Scale bar: 40µm\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/7e0d02732850b4c10d28b85f.png"},{"id":91963576,"identity":"c40030f7-e6b4-4b8d-aed4-603dca8cfddd","added_by":"auto","created_at":"2025-09-23 08:07:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":60019,"visible":true,"origin":"","legend":"\u003cp\u003eReal-time fluorescence kinetics in symbiotic regions\u003c/p\u003e\n\u003cp\u003e(A) Experimental design: FM4-64 application to distal hyphal tips. (B) Kinetics of \u003cem\u003eG. versiforme \u003c/em\u003e(red). (C)\u003cem\u003e \u003c/em\u003eKinetics of \u003cem\u003eR. irregularis\u003c/em\u003e (blue). Data are presented as mean ± SD (n = 3).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/b76bba7255f08ec6f431eaf1.png"},{"id":91963571,"identity":"fc987eda-4677-436c-b6b3-45243f7d59a9","added_by":"auto","created_at":"2025-09-23 08:07:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":44491,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of hyphal transport capacity between \u003cem\u003eGlomus versiforme\u003c/em\u003e (red) and \u003cem\u003eRhizophagus irregularis\u003c/em\u003e (blue) across time points.\u003c/p\u003e\n\u003cp\u003eData represent mean intensity ± SD (n = 3 biological replicates); Statistical significance: p \u0026lt; 0.05 (*), p \u0026lt; 0.01 (**), p \u0026gt; 0.05 not significant (ns).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/e57f422a24d8b351f20ecc4d.png"},{"id":97248509,"identity":"783dfc5c-8d5d-476d-91ff-d485eab6b9ff","added_by":"auto","created_at":"2025-12-02 13:02:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3648264,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7568699/v1/de7d3719-4eed-4834-8a8c-174e5d1649af.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Real-time fluorescence imaging platform for high-throughput screening of arbuscular mycorrhizal fungi enhancing plant nutrient uptake","fulltext":[{"header":"Background","content":"\u003cp\u003eArbuscular mycorrhizal fungi (AMF) form mutualistic symbioses with approximately 80% of land plant species, playing a crucial role in enhancing host mineral nutrient acquisition \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. This obligate symbiotic relationship is based on a bidirectional exchange: plants provide carbon sources such as fatty acids and sugars to the fungi, while AMF extend the root system via their hyphae networks \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e and secrete metabolites including proteases and siderophores, which facilitate the mobilization of organic nitrogen and metallic nutrients\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Moreover, AMF hyphae recruit beneficial rhizosphere microbes (e.g., Pseudomonas, Bacillus) through carbon-rich exudates, establishing synergistic interactions that further improve plant uptake of phosphorus, nitrogen, and micronutrients \u003csup\u003e[\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe primary site of nutrient exchange is the arbuscule\u0026mdash;a highly branched structure formed within root cortical cells that greatly expands the symbiotic interface \u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Efficient nutrient transport relies on cytoplasmic streaming within the hyphae, which enables both intrahyphal diffusion and long-distance translocation of nutrients \u003csup\u003e[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e Upon arrival at the arbuscules, nutrients are released across the periarbuscular membrane for plant assimilation \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, underscoring the fundamental role of cytoplasmic streaming in symbiotic efficiency \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eConventional methods for studying AMF symbiosis, such as field trials and compartmented cultivation systems, face significant limitations. These approaches typically depend on endpoint measurements of nutrient concentrations in plant tissues, thereby failing to capture the dynamic nature of nutrient transport processes \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Moreover, non-axenic conditions increase the risk of microbial contamination, and soil variability often compromises experimental reproducibility \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. For perennial plants, these methods can require months to years to yield results, greatly restricting the capacity for high-throughput strain screening.\u003c/p\u003e\u003cp\u003eTo address these constra challenges, we developed micro-chamber system integrated with real-time fluorescence monitoring. This platform allows for: (1) sterile co-culture, eliminating microbial contamination; (2) in situ tracking of nutrient fluxes using fluorescent tracers; and (3) quantitative analysis of uptake kinetics through spatiotemporal fluorescence profiling. Beyond providing mechanistic insights into AMF-mediated nutrient acquisition, this system offers a standardized framework for the high-throughput phenotyping of AMF strains with superior transport efficiency. This advancement supports ongoing efforts to improve crop nutrient-use efficiency and promote sustainable agriculture practices \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant Material and Hairy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRoot\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eInduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ensure consistent biomass production，transgenic hairy roots were induced in carrot (\u003cem\u003eDaucus carota\u003c/em\u003e L.) explants using \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e strain ACCC10060 \u003csup\u003e[25]\u003c/sup\u003e. These axenic hairy roots were subsequently co-cultivated with surface-sterilized AMF spores to establish symbiotic samples. The procedure involved the following steps:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e Inoculum:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(i) A single bacterial colony was inoculated into YEB broth (Yeast Extract Broth; Coolaber,\u0026nbsp;Beijing, China) and cultured at 28\u0026deg;C with agitation\u0026nbsp;200 rpm until an OD\u003csub\u003e600\u003c/sub\u003e of 1.0 was reached.\u003c/p\u003e\n\u003cp\u003e(ii) A 2 mL aliquot of this pre-culture was transferred to 100 mL of fresh YEB broth and incubated under identical the same conditions until the OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003ereached 0.6.\u003c/p\u003e\n\u003cp\u003e(iii) Spores were pelleted by centrifugation at 3,000 \u0026times; g for 3 min at 25\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e(iv) The pellet was resuspended in MS liquid medium supplemented with 100 \u0026mu;M acetosyringone (Solarbio, Beijing, China ; dissolved in DMSO) to prepare the inoculum.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHairy Root Induction:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCarrot (\u003cem\u003eDaucus carota\u003c/em\u003e L.) taproots were subjected to sequential surface sterilization as follows:\u003c/p\u003e\n\u003cp\u003e(i) Rinsing with distilled water.\u003c/p\u003e\n\u003cp\u003e(ii) Removal of epidermal tissues.\u003c/p\u003e\n\u003cp\u003e(iii) Immersion in 75% (v/v) ethanol for 30 seconds.\u003c/p\u003e\n\u003cp\u003e(iv) Treatment with 1% (v/v) sodium hypochlorite for 10\u0026ndash;20 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-culture:\u0026nbsp;\u003c/strong\u003eExplants were co-cultured for\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e3 days in darkness at 25\u0026ndash;28\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eDecontamination: Using Medium I (1% [w/v] agar supplemented with 400 mg/L Timentin; Coolaber, Beijing, China). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubculture:\u0026nbsp;\u003c/strong\u003eRoots were maintained on hormone-free full-strength MS medium (hopebiol, Qingdao, China) at 26 \u0026plusmn; 1\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAM Fungal Spore Isolation and Sterilization:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndividual spores of \u003cem\u003eGlomus versiforme\u003c/em\u003e and \u003cem\u003eRhizophagus irregularis\u003c/em\u003e were isolated from soil samples using a micropipette puller (Model P-97, Sutter Instrument, Novato, CA, USA). Surface sterilization was performed as follows:\u003c/p\u003e\n\u003cp\u003e1. Ultrasonic cleaning: 40 kHz, 100 W, for 2 minutes in sterile water (KQ-3200DE, Kunshan Ultrasonic Instruments, Jiangsu, China).\u003c/p\u003e\n\u003cp\u003e2. Chemical sterilization: 10 minutes in Solution A (2% [w/v] chloramine-T (Coolaber, Beijing, China) + 0.01% [v/v] Tween-20 (Coolaber, Beijing, China)).\u003c/p\u003e\n\u003cp\u003e3. Antibiotic treatment: 10 minutes in Solution B (200 \u0026mu;g/mL streptomycin + 100 \u0026mu;g/mL gentamicin (both from Solarbio, Beijing, China)).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSterilized spores were rinsed five times with sterile water prior to inoculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSymbiotic Co-culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 20 surface-sterilized spores were inoculated 1\u0026ndash;2 cm from carrot hairy roots in modified Strullu-Romand (MSR) agar medium\u003csup\u003e[26]\u003c/sup\u003e. Chambers were sealed and incubated at 25\u0026ndash;28\u0026deg;C in darkness. Symbiosis was confirmed at 40\u0026ndash;60 days post-inoculation (dpi) by observing hyphal penetration under an Olympus BX63 microscope (Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSymbiotic Micro-chamber Construction:\u003cbr\u003e\u003c/strong\u003eA sealed microfluidic culture chamber was constructed using quartz glass (Fig. 3), consisting of custom-made hollow quartz plates and cover slips. The chamber has overall external dimensions of 70 \u0026times; 70 \u0026times; 5 mm (L \u0026times; W \u0026times; H) and an internal cavity measuring 40 \u0026times; 40 \u0026times; 5 mm. Two centrally located alignment grooves (1 \u0026times; 1 \u0026times; 5 mm) along opposite edges of the cavity allow precise compartmentalization. A sterile nylon membrane (30 \u0026micro;m pore size; UV-treated and permeable only to AMF hyphae) divides the chamber into separate plant and hyphal compartments. Cover slips (50 \u0026times; 50 mm) were sealed to the chamber base using high-vacuum silicone grease (Dow Corning, Midland, MI, USA), followed by autoclave sterilization (121\u0026deg;C, 30 min, 15 psi) to ensure sterility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence Detection System:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed circuit design and noise analysis are provided in Supplementary Fig. S1\u003c/p\u003e\n\u003cp\u003eA high-sensitivity transimpedance amplifier (TIA) circuit (Fig.4) was developed for detecting weak bioluminescent signals. The system consists of three optimized subsystems:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1. Power Filtering:\u0026nbsp;\u003c/strong\u003eA symmetrical power filtering network using parallel 100 nF ceramic capacitors (X7R) and 10 \u0026mu;F tantalum capacitors per supply rail (\u0026plusmn;12.5 V to \u0026plusmn;18 V) to suppress multi-frequency power noise, achieving \u0026mu;V-level ripple essential for nanoampere-level signal integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Photodiode:\u003c/strong\u003e A custom photodiode operating at zero reverse bias (Vr = 0 V) with a junction capacitance (Cd) of 150 pF and shunt resistance (Rsh) of 600 M\u0026Omega;, eliminating dark current-induced shot noise while maximizing photon collection area for a peak photocurrent (Ip) of 100 nA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. TIA Core:\u003c/strong\u003e A single-stage TIA utilizing an AD795 operational amplifier with ultra-low input voltage noise (1.2 nV/\u0026radic;Hz). A 100 M\u0026Omega; thin-film feedback resistor (Rf) provides a gain of 10⁸ V/A, A strategically selected 1.3 pF compensation capacitor (Cf) stabilizes the high-impedance node by damping the resonance peak (Q = 0.453), extending the bandwidth to 3.26 kHz\u0026mdash;significantly beyond the theoretical limit of 1/(2\u0026pi;RfCd) \u0026asymp; 10.6 Hz\u0026mdash;without compromising the 190 \u0026mu;s rise time. The total output noise is 164 \u0026mu;V RMS (1.64 pA RMS referred to input), predominantly contributed by the op-amp\u0026rsquo;s voltage noise (94.5%). The design achieves an SNR of 86.6 dB (ENOB = 14.1 bits), enabling reliable detection of sub-picoampere bioluminescent signals within the target bandwidth of 1 kHz.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptoelectronic assembly\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fluorescence detection module (Fig. 5) integrates four components: an X-Cite 120Q excitation lamp (Exelitas Technologies), an excitation filter (Ex-filter; #86-977 , Edmund), an emission filter (Em-filter; #86-979 , Edmund), and a photodiode(S1336-44BQ, Hamamatsu Photonics, Hamamatsu, Japan); data acquisition was performed using an NI USB-6210 interface (National Instruments, Austin, TX, USA). In this configuration, the excitation source and Ex-filter are positioned above the plant compartment, while the Em-filter and photodiode are optically aligned below. The excitation source delivers targeted illumination to the plant samples.When fluorophore-labeled nutrients enter the detection zone, they emit fluorescence upon excitation. The emitted light passes through the Em-filter to removal stray light before being converted into photocurrent by the photodiode.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence Detection Procedure:\u003cbr\u003e\u003c/strong\u003eCarrot hairy roots cultured in-house and surface-sterilized AMF spores were used in the experiments. Hairy roots were inoculated in the plant compartment and spores in the hyphal compartment, both containing modified Strullu-Romand (MSR) medium. Sealed microchambers were incubated in darkness at 25-28\u0026deg;C. After AMF hyphal germination and penetration through the nylon membrane to establish symbiosis, co-culture continued for 40-60 days until extensive hyphal networks developed. The upper lid was gently removed, and FM4-64 fluorescent dye was applied to the hyphal compartment to label hyphae for cytoplasmic streaming quantification. The root-hyphal junction was positioned over the photodiode detector and enclosed within the detection module. Excitation was then initiated for real-time monitoring of fluorescence dynamics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Acquisition and Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe photodiode converted low-amplitude fluorescence signals into current signals. Which were processed by a transimpedance amplifier (TIA) and filter, yielding analog voltage signals. These were digitized at 250 kS/s using an via an NI USB-6210 data acquisition interface (National Instruments). Raw data were collected with a custom LabVIEW program.\u003c/p\u003e\n\u003cp\u003eData acquisition was performed at 250 kS/s using LabVIEW 2023 (National Instruments). Fluorescence kinetics were analyzed using custom MATLAB scripts (MathWorks, Natick, MA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis and Reproducibility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; SD of three biological replicates. Significance was determined using two-tailed t-test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u003cem\u003e\u0026nbsp;p\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) .\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eReal-Time Visualization of Cytoplasmic Streaming Dynamics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtraradical hyphae of arbuscular mycorrhizal fungi (AMF) form extensive networks that facilitate nutrient acquisition through cytoplasmic streaming to arbuscules \u003csup\u003e[27]\u003c/sup\u003e. However, \u003cem\u003ein situ\u003c/em\u003e monitoring of nutrient flux remains challenging due to soil heterogeneity. To address this, we developed a real-time imaging platform using the membrane-selective fluorophore FM4-64 (10 \u0026mu;M; Fig. 6). This dye effectively labeled live root tissues, intraradical hyphae, and spores within sterile micro-chambers. Using fluorescence microscopy (Olympus BX63; 560/640 nm, 5 fps), we quantified directional transport of fluorescent signals, establishing cytoplasmic streaming as a direct proxy for nutrient flux.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain-Specific Kinetics of Cytoplasmic Streaming\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur imaging platform tracked the transport of FM4-64 from distal hyphal tips (applied 1 cm from detection zones) to symbiotic interfaces (Fig. 7). Quantitative analysis revealed distinct kinetics profiles between AMF strains:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGlomus versiforme\u003c/em\u003e exhibited rapid fluorescence accumulation (200\u0026ndash;500 s: \u003cem\u003eslope\u003c/em\u003e = 0.01506 \u0026plusmn; 0.0008\u0026Delta;F/F₀\u0026middot;s⁻\u0026sup1;, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01), reaching a plateau at 500 s (\u003cem\u003epeak\u003c/em\u003e \u0026Delta;F/F₀ = 0.0007 \u0026plusmn; 0.0027).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRhizophagus irregularis\u003c/em\u003e showed a sustained increases until 800 s (\u003cem\u003eslope\u003c/em\u003e = 0.0052 \u0026plusmn; 0.0003 \u0026Delta;F/F₀\u0026middot;s⁻\u0026sup1;), with a lower peak intensity (\u0026Delta;F/F₀ = 0.0014 \u0026plusmn; 0.0007; \u003cem\u003ep\u003c/em\u003e = 0.008 vs. \u003cem\u003eG. versiforme\u003c/em\u003e; Fig. 7C).These findings indicate efficient tracer delivery to symbiotic regions, with \u003cem\u003eG\u003c/em\u003e. \u003cem\u003eversiforme\u003c/em\u003e demonstrating superior transport kinetics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Comparison of Hyphal Transport Capacity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe quantified interspecific variation by analyzing fluorescence dynamics over a 12 minute period (Fig. 8):\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eG. versiforme\u003c/em\u003e achieved a 2.3-fold higher initial transport rate (3\u0026ndash;6 min: 3.131 \u0026plusmn; 0.864 vs. 1.311 \u0026plusmn; 0.305 \u0026Delta;F/F₀\u0026middot;min⁻\u0026sup1; in R. \u003cem\u003eirregularis\u003c/em\u003e, p \u0026lt; 0.05) and reached peak intensity at 9 min (\u0026Delta;F/F₀ = 4.645 \u0026plusmn; 0.465).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eR. irregularis\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eexhibited a delayed peak at 12 min (\u0026Delta;F/F₀ = 4.029 \u0026plusmn; 0.238; not significant vs. \u003cem\u003eG. versiforme\u003c/em\u003e after 9 min; Fig. 8).\u003c/p\u003e\n\u003cp\u003eStatistical analysis (n = 3 biological replicates) confirmed the superior transport capacity of \u003cem\u003eG. versiforme\u003c/em\u003e (p \u0026lt; 0.05 at 6 and 9 min), highlighting functional divergence in AMF symbiotic efficiency.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur compartmentalized microchamber system overcomes the limitations of traditional pot cultures by enabling real-time monitoring of nutrient dynamics at the hypha-root interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Key advances include:\u003c/p\u003e\u003cp\u003e(1) \u003cb\u003eSterility-controlled co-culture\u003c/b\u003e, which eliminates artifacts from soil sterilization and ensures specific colonization by AMF;\u003c/p\u003e\u003cp\u003e(2) \u003cb\u003eIntegrated optoelectronic sensors\u003c/b\u003e that capture continuous nutrient fluxes, revealing kinetic bottlenecks undetectable with endpoint assays.\u003c/p\u003e\u003cp\u003eAlthough this platform allows precise manipulation of plant-fungal interactions, its current simplified representation of the rhizosphere microbiome does not account for essential cross-kingdom interactions. For example, bacterial communities influenced by AMF-derived strigolactones may enhance symbiosis efficiency through auxin signaling or siderophore production. Future designs incorporating modular bacterial chambers will be crucial for elucidating such tripartite interactions\u003csup\u003e[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eConstraints in Probe diversity also limit the scope of detection. Existing fluorescent target only major minerals (e.g., Pi, NH₄⁺), leaving trace elements (e.g., Zn\u0026sup2;⁺, Cu\u0026sup2;⁺) unmonitored. Integrating dynamic flux measurements with multi-omics approaches\u0026mdash;such as spatially resolved metabolomics of arbusculated cells\u0026mdash;could uncover mechanisms governing nutrient exchange across biological scales\u003csup\u003e[\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo bridge the gap between laboratory and field conditions, we propose a tiered validation pipeline:\u003c/p\u003e\u003cp\u003e(1) High-throughput screening of up to 200 AMF strains per experiment using microchambers;\u003c/p\u003e\u003cp\u003e(2) Controlled-environment validation with defined substrates that simulate soil heterogeneity;\u003c/p\u003e\u003cp\u003e(3) Field assessment via rhizoboxes to quantify functional stability under realistic agricultural conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis framework balances screening efficiency with ecological relevance, accelerating the development of beneficial AMF strains into sustainable inoculants.\u003c/p\u003e\u003cp\u003eIn summary, our system provides a transformative platform for analyzing arbuscular mycorrhizal symbiosis dynamics. By combining real-time sensing with scalable validation, it facilitates the translation of AMF research from mechanistic insights to practical applications, supporting advances in microbiome engineering for sustainable agriculture.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe developed a real-time fluorescence imaging platform incorporating photodiode arrays to dynamically quantify nutrient flux at plant-AMF symbiotic interfaces. This system enables high-throughput functional classification of arbuscular mycorrhizal fungi (AMF) strains by monitoring fluorescence kinetics\u0026mdash;such as FM4-64 tracer accumulation rates\u0026mdash;achieving a tenfold increase in screening speed compared to conventional field-based methods. Our approach effectively connects mechanistic research with sustainable agriculture, promoting targeted AMF breeding and microbiome engineering.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eAMF\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eArbuscular mycorrhizal fungi\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eYEB\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eYeast Extract Broth\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eMSR\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStrullu-Romand medium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTIA\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etransimpedance amplifier\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the China Huanghuai Sea High-yield Key Cultivation Technology Innovation and Integration Demonstration (Grant No. 2023YFD2300204), the National Natural Science Foundation of China (Grant No. 32401739) and the China National Modern Agricultural Production Technology System(CARS-03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e1. Agronomy College, Henan Agriculture University/ State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Zhengzhou 450046, Henan, China\u003c/p\u003e\n\u003cp\u003e2. National Engineering Research Centre for Wheat, Zhengzhou 450046, Henan, China\u003c/p\u003e\n\u003cp\u003eYonghua Wang, Bo Yu, Tiancai Guo, GuangLe Zhang, Lixue Yuan, Yongxin Zhang, Xinyuan Zhang, Li Zhang, Ruxue Chen\u003c/p\u003e\n\u003cp\u003e3. College of Resources and Environment, Henan Agricultural University, Zhengzhou 450046, Henan, China\u003c/p\u003e\n\u003cp\u003eZhuangzhuang Wang\u003c/p\u003e\n\u003cp\u003e4. Department of Crop and Soil Sciences of North Carolina State University, Raleigh, 27695, North Carolina, US\u003c/p\u003e\n\u003cp\u003eXiaohang Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB. Y. and G.L. Z. designed and fabricated the circuit board, collected and analyzed data, and drafted the manuscript. B. Y., Y.H. W. and T.C. G. conceived and designed the study, developed the methodology, and revised the manuscript substantively. L.X. Y. and Y.X. Z. contributed to data acquisition, analysis, and interpretation; X.H. W., X. Y. Z., L. Z., R.X. C., and Z.Z. W. contributed to data acquisition and collation; All authors read and approved the final manuscript and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Bo Yu and Yonghua Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOldroyd GED, Leyser O. 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The Promises, Challenges, and Opportunities of Omics for Studying the Plant Holobiont. Microorganisms. 2022;10(10). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms10102013\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms10102013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Figure","content":"\u003cp\u003eSupplementary Figure S1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"arbuscular mycorrhizal fungi, nutrient transport, cytoplasmic streaming, high-throughput screening, fluorescence imaging","lastPublishedDoi":"10.21203/rs.3.rs-7568699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7568699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eArbuscular mycorrhizal fungi (AMF) are ancient soil symbionts that form mutualistic associations with approximately 80% of terrestrial plant species. They enhance host nutrient and water acquisition in exchange for photosynthetic carbon. Current AMF research relies on field trials, compartmented cultivation and pot cultures‒methods that are time-consuming (months to years) and unable to monitor dynamic nutrient transport, thus limiting efficient strain screening.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe developed a real-time fluorescence imaging platform integrating sterile symbiotic microchambers with photodiode array detection. This system enables non-invasive, quantitative tracking of nutrient flux at plant-fungal interface. Distinct AMF strains exhibit significant differences in fluorescence kinetics\u0026mdash;such as accumulation rate and peak intensity\u0026mdash;providing measurable indicators of transport efficiency. The platform allows high-throughput functional screening of AMF strains, dramatically accelerating the identification of high-performance symbionts.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur method overcome the temporal and technical limitations of conventional AMF screening approaches. By enabling simultaneous real-time monitoring and high-throughput analysis, it shortens screening cycles and establishes a standardized framework for (1) precision breeding of efficiency AMF strains, (2) mechanistic study of nutrient exchange, and (3) development of sustainable microbial inoculants.\u003c/p\u003e","manuscriptTitle":"Real-time fluorescence imaging platform for high-throughput screening of arbuscular mycorrhizal fungi enhancing plant nutrient uptake","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 08:07:17","doi":"10.21203/rs.3.rs-7568699/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89ef0912-2011-4045-aa1d-bd151a956091","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-13T10:47:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 08:07:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7568699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7568699","identity":"rs-7568699","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}